3

Electronic Supplementary Material

Polymerase chain reactions (PCR)

All PCR reactions contained 1 ml of template DNA (~150 ng), 3 ml of reaction buffer (Promega), 3.6 ml of 25 mM MgCl2 (i.e. 3 mM MgCl2), 6 ml of dNTP mixture containing 125 mM of each dNTP, 1.2 ml of each primer (5 mM dilutions), 1 unit of Taq DNA polymerase (Promega) and water to a final volume of 30 ml. PCR profiles consisted of an initial denaturing step (94oC for 3 min), 35 cycles of denaturing (94oC for 30 s), annealing for 45 s at a primer-specific annealing temperature (Table 1) and extension (72oC for 45 s), and a final extension step (72oC for 10 min). PCR products were purified using the UltraCleanTM15 DNA Purification Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA), sequenced in both directions using Big Dye terminator version 3.1 (Applied Biosystems) and run on a 3130xl Genetic Analyser (Applied Biosystems) according to the manufacturer’s instructions.

Sequence alignment

The nuclear genes could readily be aligned by eye, but as the alignment of the S7 intron was challenging, we used the program BAli-Phy 2.0.2 (Suchard & Redelings 2006) (a program that simultaneously estimates phylogeny and alignment) to generate a consensus-75 alignments. We specified 10 000 iterations (with a burn-in of 10%) and applied the GTR+I+G model (Rodríguez et al. 1990). The process was repeated three times to check for consistency of results, and poorly aligned sites were then removed using the GBlocks (Talavera & Castresana) server available at http://molevol.cmima.csic.es/castresana/Gblocks_server.html (the most stringent conditions were specified). The DNA sequence data-set used in all analyses included 1476 bp of RAG1, 711 bp of myh6, 447 bp of Rhodopsin, 459 bp of Tmo4c4 and 293 bp of S7 (Table 2).

Molecular dating

Molecular dating was performed using the program Beast 1.4.8 (Drummond & Rambaut 2007). An uncorrelated lognormal relaxed clock was specified. Sequences were partitioned as described for the phylogenetic analyses, and all nodes that were strongly supported in the phylogenetic analyses (bootstrap values > 75% and posterior probabilites >95%) were constrained to be monophyletic. A Yule process was used as a tree prior. A chain length of 20 million generations was specified, and parameters were saved every 200 generations. Results were checked in Tracer (available at beast.bio.ed.ac.uk/Tracer) to ensure that effective samples sizes for all parameters were above 200 and that posterior probabilities remained constant after a burn-in of 10%. To check for consistency of results, the analyses were repeated three times for each of two methods of calibrating the tree. A chronogram for one of the methods (using three calibration points) was then produced using the programs TreeAnnotator 1.4.6 (available at http://beast.bio.ed.ac.uk/TreeAnnotator) and FigTree 1.1 (available at http://beast.bio.ed.ac.uk/FigTree)

Table 1. Primers used to amplify five nuclear loci in seahorses and other syngnathids.
Locus / Primer Name / Primer Sequence / Annealing
Temp. (oC) / Reference
RAG1 / RAG1-2510Fa
RAG1-4090Ra
RAG1-2533Fb
RAG1-4078Rb / TGGCCATCCGGGTMAACAC
CTGAGTCCTTGTGAGCTTCCATRAAYTT
CTGAGCTGCAGTCAGTACCATAAGATGT
TGAGCCTCCATGAACTTCTGAAGRTAYTT / 55
55 / Li & Ortí 2007
Lopez et al. 2004
Lopez et al. 2004
Lopez et al. 2004
myh6 / myh6_F459a
myh6_R1325a
myh6_F507b
myh6_R1322b / CATMTTYTCCATCTCAGATAATGC
ATTCTCACCACCATCCAGTTGAA
GGAGAATCARTCKGTGCTCATCA
CTCACCACCATCCAGTTGAACAT / 50
60 / Li et al. 2007
Li et al. 2007
Li et al. 2007
Li et al. 2007
Rhodopsin / Rod-F2Ba
Rod-5Ra
Rod-F2Wb
Rod-R4nb / GTCTGCAAGCCCATCAGCAACTTCCG
GGTGGTGATCATGCAGTGGCGGAA
AGCAACTTCCGCTTCGGTGAGAA
GGAACTGCTTGTTCATGCAGATGTAGAT / 62
56 / Sevilla et al. 2007
Sevilla et al. 2007
Sevilla et al. 2007
Sevilla et al. 2007
Tmo4c4 / Tmo4c4F
Tmo4c4R / CCTCCGGCCTTCCTAAAACCTCTC
CATCGTGCTCCTGGGTGACAAAGT / 55 / Streelman & Karl 1997, Streelman et al. 1998
S7 / S7RPEX1F
S7RPEX2R / TGGCCTCTTCCTTGGCCGTC
AACTCGTCTGGCTTTTCGCC / 55 / Chow & Hazama 1998
Chow & Hazama 1998
aPrimers for first round PCR; bPrimers for second round PCR using 1 in 100 dilutions of first round PCR products as template
Table 2. Syngnathid specimens used for the phylogeny, and their museum collection numbers and GenBank accession numbers.
Species / Collection No. / GenBank Accession Numbers
RAG1 / myh6 / Rhodopsin / Tmo4c4 / S7
Hippocampus reidi / RM2170 / FJ905768 / FJ905779 / FJ905790 / FJ905801 / DQ288386
H. ingens / RM2530 / FJ905769 / FJ905780 / FJ905791 / FJ905802 / DQ288382
H. zosterae / RM2310 / FJ905770 / FJ905781 / - / FJ905803 / DQ288379
H. breviceps / (own collection) / FJ905771 / - / FJ905792 / FJ905804 / AY277320
H. bargibanti / (own collection) / FJ905772 / FJ905782 / FJ905793 / - / AY277309
Idiotropiscis lumnitzeri / (own collection) / FJ905773 / FJ905783 / FJ905794 / FJ905805 / FJ905808
Hippichthys penicillus / I.41864-016 / FJ905774 / FJ905784 / FJ905795 / FJ905806 / FJ905809
Vanacampus margaritifer / I.41084-042 / FJ905777 / FJ905787 / FJ905798 / - / FJ905811
Corythoichthys sp. / SAIAB 80704 / - / FJ905788 / FJ905799 / FJ905807 / -
Stigmatopora nigra / I.43656-001 / FJ905776 / FJ905786 / FJ905797 / - / FJ905810
Syngnathus temminckii / (own collection) / FJ905775 / FJ905785 / FJ905796 / - / AY277308
Doryrhamphus excisus / SAIAB 78058 / FJ905778 / FJ905789 / FJ905800 / - / FJ905812
RM = Redpath Museum, Montreal; I = Australian Museum, Sydney; SAIAB = South African Institute of Aquatic Biodiversity, Grahamstown, South Africa; RAG1 = recombination activating gene 1, myh6 = myosin heavy chain 6, S7 = first intron of the S7 ribosomal protein

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